| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 97 === TFP1 is an immunomudulatory protein previously purified from jelly fungi (Tremella fuciformis Berk). The objectives of this study was to clarify the biochemical characteristics of TFP1, to further investigate its regulating function and its activation pathway on mouse peritoneal macrophages as well as to construct its yeast expression system. Mass spectrometry analyses obtained the molecular weight of native TFP1 (11.0 kDa), agreeing its genetically translated value (11.3 kDa). Based on gel-filtration and SDS-PAGE analyses, the recombinant TFP1 showed a molecular mass of 24 kDa, suggesting that TFP1 could be a homodimer protein. In addition, TFP1 activated mouse peritoneal macrophages, increased the production of TNF-a and IL-1b and enhanced the mRNA expression of TNF-a, IL-1b, IL-6, IL-12p35, IL-12p40, CCL3, and CXCL10. Flow cytometry analysis demonstrated that TFP1 increased the phagocytosis activity and CD86 expression by the cells. Moreover, EMSA result showed a TFP1-induced activation of the transcription factor NF-kB. The stimulatory effects of TFP1-promoted TNF-a secretion on macrophages were inhibited under antibody neutralization and TLR4 deficiency (TLR4-/-). Based on these results, TLR2/TLR4 signaling pathway was suggested to be involved in TFP1-induced NF-kB activation. Furthermore, TFP1 gene was cloned into pYEX-S1 vector and expressed by Saccharomyces cerevisiae to produce recombinant TFP1 and His-tagged TFP1. This study clarified the mechanism and activities of TFP1 on mouse peritoneal macrophages and also provided more information on the application of feed and food utilization.
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